Molecular characterization of 21 X-ALD Portuguese families: identification of eight novel mutations in the ABCD1 gene CarlaP.Guimar~ aes, a,b Manuela Lemos, a,c ClaraS a-Miranda, a,c andJorgeE.Azevedo a,b, * a Department of Genetic Neurobiology, IBMC, Porto University, Rua do Campo Alegre, 823, Porto 4150-180, Portugal b ICBAS-Porto University, Porto, Portugal c Enzymology Unit, IGMJM, Porto, Portugal Received 22 January 2002; received in revised form 27 February 2002; accepted 28 February 2002 Abstract X-linked adrenoleukodystrophy (X-ALD) is the most common inherited peroxisomal disorder. The gene associated with X-ALD, ABCD1, encodes a peroxisomal ATP-binding cassette half-transporter. In this study, we describe the molecular charac- terizationof21affectedPortuguesefamilies.Thecompletecodingregionofthe ABCD1genewasamplifiedbyreversetranscription polymerasechainreaction(RT-PCR)orgenomicPCR.Afterconformation-sensitivegelelectrophoresisanalysis,fragmentswitha conformational heteroduplex pattern were sequenced. Using this strategy, we have identified 14 missense mutations, two nonsense mutations,twosplicingsitedefects,andthreesmalldeletions,twoofthemresultinginframeshifts.Eightofthegeneticalterations characterizedinthisstudyarenovel.Thelevelsofthe ABCD1transcriptaswellasthelevelsofALDPinculturedskinfibroblastsof maleprobandswerealsodeterminedinmostcases.Thelevelsofthe ABCD1transcriptinonepatient(correspondingtoanonsense mutation) were below the detection limit of Northern-blotting analysis. ALDP was found at normal levels in only three patients, absent in five (corresponding to a double missense, two nonsense, and two frameshift mutations), and decreased in all the others. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: X-ALD; ABCD1 gene; Mutations; Peroxisome disorders 1. Introduction The adrenoleukodystrophy (X-ALD, OMIM #300100) is an inborn X chromosome linked disease. This neurodegenerative disorder is characterized by greatclinicalexpressionvariability.Accordingtotheage ofonset,theorgansaffectedandtherateofprogression of neurologic symptoms seven phenotypes can be considered in males: childhood cerebral form (CCER), adolescent cerebral ALD, adrenomyeloneuropathy (AMN), adult cerebral ALD, Addison disease only (AO),olivo-ponto-cerebellar,andasymptomatic.CCER isthemostseverephenotypewithanonsetat3–10years ofage;afterthefirstsymptomsappearthecourseofthe disease is rapidly progressive leading to death. The ad- renomyeloneuropathy phenotype has an onset in young adulthood. AMN patients who have a normal brain MRI with neurological involvement confined to the spinalcordandperipheralnervesarereferredtoas‘pure AMN.’However,almost40%oftheAMNpatientshave ordevelopcerebraldisabilitybeingreferredtoasAMN- cerebral. The Addison-only X-ALD form is character- ized by adrenocortical insufficiency without nervous systeminvolvement.However,thesepatientsareathigh risktodevelopneurologicsymptoms(forreviewsee[1]). The main biochemical abnormality associated with X-ALD is the accumulation of unbranched saturated very long chain fatty acids (VLCFA) in plasma and tissues [2]. This is due to an impaired capacity of the peroxisome to metabolize these fatty acids via b-oxida- tion [3]. The increase in the VLCFA levels provides a reliable diagnostic tool for prenatal and postnatal identification of affected males [1]. Most carrier women can also be identified using this criterion [4]. The ALD gene (presently known as ABCD1 gene) was identified by positional cloning in 1993 [5]. It Molecular Genetics and Metabolism 76 (2002) 62–67 www.academicpress.com * Corresponding author. Fax: +351-226092404. E-mail address: jazevedo@ibmc.up.pt (J.E. Azevedo). 1096-7192/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII:S1096-7192(02)00023-9